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Abstract Objectives This study was performed in the frame of a more extensive study dedicated to the integrated analysis of the single-cell transcriptome and chromatin accessibility datasets of peripheral blood mononuclear cells (PBMCs) with a large-scale GWAS of 45 complex traits in Chinese Holstein cattle. Lipopolysaccharide (LPS) is a crucial mediator of chronic inflammation to modulate immune responses. PBMCs include primary T and B cells, natural killer (NK) cells, monocytes (Mono), and dendritic cells (DC). How LPS stimulates PBMCs at the single-cell level in dairy cattle remains largely unknown. Data description We sequenced 30,756 estimated single cells and mapped 26,141 of them (96.05%) with approximately 60,075 mapped reads per cell after quality control for four whole-blood treatments (no, 2 h, 4 h, and 8 h LPS) by single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq). Finally, 7,107 (no), 9,174 (2 h), 6,741 (4 h), and 3,119 (8 h) cells were generated with ~ 15,000 total genes in the whole population. Therefore, the single-cell transcriptome and chromatin accessibility datasets in this study enable a further understanding of the cell types and functions of PBMCs and their responses to LPS stimulation in vitro.